The goal of many chemical analysis protocols is to separate a sample (blood, tears, urine, water from a well, etc.) into its individual components or constituents so that each component may be evaluated without any interference from other components. One technique that is often employed to separate various constituents of a sample from each other is chromatography, where liquid chromatography (“LC”) is often employed. Liquid chromatography is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a liquid or solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column. Chromatography may be coupled with a suitable detection system that can characterize each type of separated constituent. One liquid chromatography protocol that is often employed due to its versatility is high performance liquid chromatography (“HPLC”).
Generally, HPLC includes passing a sample of constituents in a high pressure fluid or solvent (called the mobile phase) through a tube or column. The column is packed with a stationary phase. The stationary phase is typically composed of a substrate such as particles, e.g., porous beads or the like. The pore sizes can be varied to allow certain sized analytes to pass through at different rates. As the constituents pass through the column they interact with the mobile and stationary phases at different rates. The difference in rates is due to the difference in one or more physical properties of the constituents, e.g., different polarities. The constituents that have the least amount of interaction with the stationary phase, or the most amount of interaction with the mobile phase, will thus exit the column faster.
As the various constituents exit the column, they can be detected by various techniques, e.g., refractive index, electrochemical, or ultraviolet-absorbance, which can indicate the presence of a constituent. The amount of constituent exiting the column may be determined by the intensity of the signal produced in a detector. A detector is employed to measure a signal peak as each constituent exits the column. By comparing the time it takes for the peak to show up (also referred to as the retention time) with the retention times for a mixture of known compounds, the constituents of unknown sample mixtures can be identified. By measuring the signal intensity (also referred to as the response) and comparing it to the response of a known amount of that particular analyte, the amount of analyte in the mixture can be determined.
One particularly useful mode of HPLC—particularly for the separation of highly polar or ionizable constituents, is reversed phase high performance liquid chromatography (“RP-HPLC”). RP-HPLC primarily operates on the basis of hydrophilicity and lipophilicity to separate various constituents of a liquid medium from each other. The stationary phase includes a substrate (which may be a plurality of particles) that has bound chemical moieties (i.e., a bonded phase), such as hydrophobic chains, e.g., bound alkyl chains, and the like, which facilitate the separation of the constituents. Accordingly, the greater the hydrophobicity of the bound chemical moieties, the greater is the tendency of the hydrophobic constituents in the mobile phase to be retained in the column while the hydrophilic constituents are eluted more rapidly from the column than the hydrophobic constituents.
Regardless of the type of liquid chromatography protocol employed, the particular mobile phase employed is important to the outcome of the protocol. For example, in order to achieve sufficient retention of certain constituents, it may be necessary to use a high aqueous mobile phase. However, when such high aqueous mobile phases are used, it is not uncommon to observe a decrease in retention of constituents over the course of the chromatography procedure, where oftentimes retention times are decreased to a point that any separation of constituents is lost.
While it is not completely clear why this loss in retention occurs when employing a high aqueous mobile phase with a stationary phase that includes hydrophobic functional groups—as is the case with RP-HPLC, it is hypothesized that the hydrophobic bonded phase (e.g., bonded alkyl chains) that is fully extended or solvated in an organic phase (e.g., 100% methanol) collapses in the highly aqueous mobile phase employed in the chromatography protocol. In other words, this behavior of retention decreasing over time in a high aqueous mobile phase is thought to be attributed to the chains of the functional groups of the stationary phase “collapsing” onto other chains and onto the surface of the particles to which they are bonded. Accordingly, this phenomenon is often referred to as “phase collapse”. When phase collapse occurs, the surface of the stationary phase is less accessible as compared to a surface where the chains are fully extended. Accordingly, when phase collapse occurs, there is less availability of the bonded phase to interact with sample constituents and consequently retention decreases.
A variety of techniques have been developed to try to combat phase collapse. One such technique that is often employed reverses the phase collapse process. This is accomplished by flooding the stationary phase with significant volumes of a high organic content mobile phase followed by quickly switching back to the high aqueous phase. However, this is not a complete solution as the retention will again decrease when the highly aqueous mobile phase is used. Another technique is to incorporate polar groups near the substrate surface which interact with the highly aqueous phase and provide a solvated surface that helps prevent phase collapse. However, this technique also has disadvantages, as retention times are typically much lower than protocols without these polar groups and the protocols for fabricating such stationary phases increases in complexity, thus increasing manufacturing costs.
Accordingly, there continues to be an interest in the development of new methods and devices for separating constituents of a highly aqueous fluid. Of particular interest is the development of such methods and devices that do not exhibit phase collapse, are easy to use and are cost effective.
REFERENCES OF INTEREST INCLUDE: J. E. O'Gara, et al., Embedded Polar Group Bonded Phases for High Performance Liquid Chromatography, LCGC, 19(6), 632 (2001); Reid, et al., Compatibility of C18 HPLC Columns with Pure Aqueous Mobile Phase, American Lab., 7, 24 (1999); Przybyciel, et al., Phase Collapse in Reversed-Phase LC, LCGC, 20(6), 516 (2002).